Placental isoferritins for the prognosis and diagnosis of immunosuppression

ABSTRACT

Methods for the diagnosis and prognosis of immunosuppressive conditions are disclosed, involving the detection of a particular isoform of ferritin, placental ferritin (PLF), in patient samples such as sera or on peripheral blood lymphocytes. PLF is elevated in immunosuppressed patients at early stages of disease; by contrast, adult insoferritins are elevated at late stages of immunodeficiency. Depending upon the nature of the disease associated with the immunodeficiency, the elevated levels of PLF detected at early stages may remain elevated or diminish as disease progresses. Examples are described in which elevated levels of PLF were detected at very early stages of of HIV-infection. The elevated levels diminished as disease progressed from ARC to AIDS. By contrast, adult isoforms of ferritin became elevated at late stages of disease.

The present application is a divisional of copending application Ser.No. 07/689,656 filed Apr. 23, 1991, now U.S. Pat. No. 5,120,640 issuedJun. 9, 1992, which is a continuation of application Ser. No. 07/164,947filed Mar. 7, 1988, which is a continuation-in-part of Ser. No.07/148,133 filed Jan. 22, 1988, now U.S. Pat. No. 4,882,270 issued Nov.21, 1989, which is a continuation of application Ser. No. 06/568,275filed Jan. 4, 1984, which is a continuation-in-part of application Ser.No. 373,715 filed Apr. 30, 1982 claiming priority under 35 U.S.C. §119to Israel application Ser. No. 62879 filed May 15, 1981, each of whichis incorporated by reference herein in its entirety.

TABLE OF CONTENTS

1. Introduction

2. Background of the Invention

3. Summary of the Invention

3.1. Definitions

4. Description of the Figures

5. Detailed Description of the Invention

5.1. Placental Ferritin and Monoclonal Antibodies Specific for PlacentalFerritin

5.1.1. The Molecular Heterogeneity of Placenta Ferritin

5.1.2. The Subunit Structure of Placenta Ferritin Reactive with H9 andG8 McAbs

5.1.3. Enzymatic Digestion of Placenta Isoferritin with V8 Protease

5.2. Detection of Placental Ferritin

5.3. Placental Ferritin and Immunosuppressive Conditions

5.3.1. Placental Ferritin and HIV Infection

6. Preparation of Monoclonal Antibodies Specific for Placental Ferritin

6.1. Preparation of Placental Ferritin

6.2. Preparation of Monoclonal Antibodies that Bind to PlacentalFerritin

6.2.1. Materials and Methods

6.2.2. Immunization Protocol

6.2.3. Spleen Cell-Myeloma Fusion

6.2.4. Screening Protocols

6.3. Preparation of Monoclonal Antibodies Specific for PlacentalFerritin and Not Cross Reactive with Normal Ferritin

6.3.1. Immunization and fusion Protocols

6.3.2. Characterization of Monoclonal Antibody CM-H9

6.4. Immunoassays for Lymphocyte-Bound Placental Ferritin Using CM-H9Monoclonal Antibody

6.4.1. Collection of Lymphocytes

6.4.2. Radioimmunoassay Procedures

6.4.3. Cytotoxic Assay Procedure

6.4.4. Results: Reactivity of CM-H9 Monoclonal Antibody with Lymphocytesin Certain Diseases

7. Isoferritins in HIV Infection: Relation to Clinical Stage, CD8⁺Lymphocyte Binding and the Pathogenesis of AIDS

7.1. Materials and Methods

7.1.1. Subjects

7.1.2. Isolation of Lymphocytes

7.1.3. Monoclonal Antibodies

7.1.4. Flow Cytometry and Immunofluorescence Staining

7.1.5. Immunofluorescence Staining of Isoferritin on LymphocyteMembranes Using CM-H9 McAb

7.1.6. Immunofluorescence Staining of Isoferritin in LymphocyteCytoplasm Using CM-H9 McAb

7.1.7. Levamisole Treatment of Mononuclear Cells

7.1.8. Quantitative Determination of Serum Isoferritin

7.1.9. Statistical Analyses

7.2. Results

7.2.1. Serum Levels of Ferritin and PLF in Patients with HIV Infection

7.2.2. Relationship of High Serum PLF and Normal Ferritin To DiseaseProgression

7.2.3. Cell Surface Antigens of Lymphocytes from HIV Infected Patients

7.2.4. The Effect of Levamisole on Cell Surface Antigens of Lymphocytesfrom HIV Infected Patients

8. Isoferritins in Patients with Lymphoproliferative Diseases

8.1. Materials and Methods

8.1.1. Subjects

8.1.2. Monoclonal Antibodies

8.1.3. Quantitative Determinations of Ferritin

8.1.4. Monoclonal Antibody ELISA For PLF and Common Isoferritins

8.2. Results

8.2.1. Evaluation of Liver Ferritin Standard by Different ELISAs

8.2.2. Binding of Placenta and Liver Ferritins to CM-G8 and CM-H9 McAbs

8.2.3. Isoferritins in the Serum of Healthy Individuals and Patientswith Lymphoproliferative Diseases

9. Isoferritins in Autoimmune Conditions

10. Deposit of Hybridoma

1. INTRODUCTION

The present invention relates to methods for the diagnosis and prognosisof immunosuppressive conditions. The method of the invention involvesthe detection of placental ferritin (PLF) in patient samples such asserum, or on peripheral blood lymphocytes. Elevated levels of PLF aredetected in patients at early stages of immunosuppression. Dependingupon the nature of the disease associated with the patients,immunosuppressed condition, the elevated PLF levels may decline withprogression of disease. The detection and measurement of PLF may beaccomplished using monoclonal antibodies described herein.

The invention is demonstrated by way of examples in which elevated PLFwas detected in sera of subjects infected with human immunodeficiencyvirus (HIV). Individuals at early stages of disease exhibited thehighest PLF levels which declined as the disease progressed.

2. BACKGROUND OF THE INVENTION

Ferritin is an iron storage protein which maintains iron in anavailable, non-toxic form. A variety of ferritin isoforms have beenisolated from different tissues. The variability of ferritincharacteristics appear to be mainly caused by the presence of differentsubunit types in the multimeric protein shell (Drysdale, 1977, CibaFound. Symp. 51:41; Arosio, et al., 1978, J. Biol. Chem. 253:4451;Watanabe et al., 1981, Biochem. Biophys. Res. Comm. 103:207). In fact,three ferritin subunits have been described. The L subunit (19 Kd),prevalent in iron loaded tissues, the H subunit (21 Kd), predominant iniron poor and malignant cells (Drysdale, 1977, supra; Arosio, 1978,supra) and the glycosylated G subunit (24 Kd) isolated from serum (Cragget al., 1981, Biochem. J. 199:565). Different isoferritins containdifferent proportions of L and H subunit types. More recently,preliminary analysis of cDNA clones revealed that the H and L subunitsare encoded by rather complex families of genes (Brown et al., 1983,Proc. Natl. Acad. Sci. USA 73:857; Costanzo et al., 1984, EMBO J. 3:23),suggesting that the heterogeneity of ferritin molecules may be evengreater than presently determined.

Various ferritin isoforms have been isolated from normal and malignanttissues, the most acidic ones predominating in tumor and fetal tissues(Drysdale, 1976, Ciba Found. Symp. 51:41; Arosio et al., 1978, J. Biol.Chem. 253:4451). It has been suggested that the assay of acidicisoferritin in the serum may be of value in the diagnosis of malignancy(Hazard et al., 1977, Nature 265:755). Elevated concentrations of serumferritin were found in patients suffering from a variety of malignantdiseases, including acute lymphocytic leukemia (ALL) (Matzner et al.,1980, Am. J. Hematol. 9:13), hepatoma (Giannoulis, 1984, Digestion30:236), breast cancer (Jacobs et al., 1976, Br. J. Cancer 34:286), andrecently Hodgkin's disease (Bezwoda et al., 1985, Scand. J. Haematol.35:505). In assays based on antibodies against HeLa cell ferritin,Hazard and Drysdale found higher concentrations of ferritin in sera frompatients with various tumors than in the same sera assayed by antibodiesdirected against normal liver ferritin (Hazard, et al., supra.). Othershave failed to demonstrate a consistent pattern of isoferritins in tumortissues (Cragg et al., 1977, Br. J. Cancer 35:635; Halliday et al.,1976, Cancer Res. 36:4486) or in sera obtained from patients with tumors(Jones et al., 1978, Clin. Chim. Acta. 85:81; Jones et al., 1980, Clin.Chim. Acta. 106:203). There are, therefore, conflicting views as to theorigin and specificity of the elevated serum ferritin in malignantdiseases.

3. SUMMARY OF THE INVENTION

The present invention is directed to a method for the diagnosis andprognosis of immunosuppressive conditions involving the detection of aparticular isoform of ferritin, placental ferritin (PLF), in patientsamples such as sera or on peripheral blood lymphocytes. The method ofthe invention is based, in part, on the surprising discovery that PLF(and not other isoferritins) is elevated in immunosuppressed patients atearly stages of disease. Depending upon the nature of immunosuppressedpatient's disease, the levels of PLF may remain elevated or may diminishas the disease advances. By contrast to PLF, adult isoferritin levelsare elevated at late stages of immunodeficiency. This discovery was madepossible, in part, by the development of monoclonal antibodies such asCM-H9 described herein (and in related parent applications) which bindto PLF exclusive of other ferritins such as the liver and spleenisoforms. These monocolonal antibodies enabled the detection ofpatients' levels of PLF exlusively, during the course of disease. Inaccordance with the invention, monoclonal antibodies exhibiting thistype of specificity can be used in immunoassays to monitor levels of PLFin patient samples. Such PLF profiles can be used in the diagnosis andprognosis of immunosuppression.

3.1. DEFINITIONS

    ______________________________________                                        AIDS =      acquired immune deficiency syndrome                               ARC =      AIDS-related complex                                               BSA =      bovine serum albumin                                               CD4 = T4 = marker of helper/inducer T lymphocytes                             CD8 = T8 = marker of cytotoxic/suppressor T                                              lymphocytes                                                        CD2 = T11 =                                                                              marker of total T cell population, sheep                                      erythrocyte rosette receptor                                       ELISA =    enzyme linked immunosorbent assay                                  HIV =      human immunodeficiency virus; HTLV-III;                                       LAV                                                                McAb =     monoclonal antibody(ies)                                           PBS =      phosphate-buffered saline                                          PLF =      placental isoferritin (also referred to                                       as oncofetal ferritin)                                             SD =       standard deviation                                                 ______________________________________                                    

4. DESCRIPTION OF THE FIGURES

FIG. 1. The mean serum levels of PLF (FIG. 1A) and normal ferritin (FIG.1B) measured simultaneously in HIV infected patients and in healthyblood bank donors. n=number of subjects tested. The bars represent mean+1 SD (standard deviation). (*) represents values significantly higherthan in blood bank donors by t-test p<0.01; xx, p<0.001.

FIG. 2. The ratio of serum PLF per CD4⁺ lymphocyte of HIV infectedpatients. The bars represent mean +1 SD.

FIG. 3. Scattergram of the percentage of circulating lymphocytespositively stained for PLF with CM-H9 McAb, in HIV infected patientsfrom categories A-E. Each point represents the determination in a singlepatient.

FIG. 4. The effect of levamisole treatment of lymphocytes from HIVinfected patients on the detectable number of T4⁺, and T8⁺ (FIG. 4A) andPLF⁺ (FIG. 4B) cells. Lymphocytes were incubated in vitro withlevamisole (40 μg/ml) or with medium (in untreated cells), for 30minutes at 37° C. prior to incubation with the conjugated McAbs.

FIG. 5. Scattergram illustrating the total serum ferritin level inpatients with hematologic malignancies (first seven columns) and inhealthy individuals (right column). Total ferritin was measured byMcELISA type A using liver ferritin as standard.

FIG. 6. Scattergram exhibiting serum PLF levels in patients withhematologic malignancies (first seven columns) and in healthyindividuals (right column). PLF was measured by McELISA type B usingplacental ferritin as standard.

FIG. 7A. Elution profile of PLF. Placental ferritins were prepared asdescribed, infra, and PLF was eluted from a DEAE-cellulose column usinga Tris-HCl (pH 7.5) gradient of 0.02M to 0.05M.

FIG. 7B. The content of PLF in each fraction as assayed by ELISA. Thefollowing capture/detection antibodies were used in ELISA sandwichassays: CM-H9 capture/CM-H9 detection; CM-G8 capture/CM-G8 detection;and CM-G8 capture/CM-H9 detection.

FIG. 8. Scattergram of PLF levels detected in patients with autoimmunediseases. PLF levels are elevated in those diseases which arecharacterized by immunosuppression.

5. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods for the diagnosis and prognosisof immunodeficient or immunosuppressive conditions which may be causedby any of a number of diseases or agents. For example, immunosuppressionwhich occurs with acquired immunodeficiency syndrome (AIDS) caused byHIV infection, certain lymphomas and leukemias as well as certainautoimmune conditions such as rheumatoid arthritis, myasthenia gravis,multiple sclerosis and the like may be diagnosed and staged using themethods of the invention.

The invention is based, in part, upon the surprising discovery that PLF(placental ferritin), as opposed to isoforms of adult ferritin, iselevated in immunosuppressed patients at early stages of disease.Depending upon the nature of the immunosuppressed patient's disease, thelevels of PLF may remain elevated or may diminish as the diseaseadvances. By contrast to PLF levels, adult ferritin is elevated at latestages of immunodeficiency. This discovery was made possible, in part,by the development of a monoclonal antibody, CM-H9 (described herein andin parent applications) which is specific for PLF and does not crossreact with adult ferritin.

In accordance with the invention, measurement of PLF levels in patientsamples can be used for the early diagnosis of immunosuppression.Moreover, the monitoring of both PLF and normal adult ferritin levelscan be used prognostically to stage the progression ofimmunosuppression. Although a variety of tissues can be tested for thepresence of PLF and/or adult ferritin, serum and peripheral bloodlymphocytes (PBL) are convenient test samples. While adult ferritin canbe assayed in serum (and in various tissues of the body) PLF occurs notonly in serum but also appears to bind to the surface of a particularsubset of circulating lymphocytes. Thus, when PLF is first produced atvery early stages of disease, the circulating lymphocyte subset may bindall the available PLF so that the serum levels of PLF may appear to benormal, while the circulating lymphocytes will test positive for PLF.However, as increasing amounts of PLF are produced during early stagesof immunosuppression, the subset of lymphocytes which bind to PLF willbecome "saturated" at which point elevated serum levels of PLF should bedetected.

Depending upon the nature of the immunosuppressed patient's disease, PLFlevels may remain elevated or may decrease as the disease progresses.For example, in HIV-infected patients, PLF levels are elevated at earlystages of disease, yet diminish at late stages of disease. By contrast,in patients with lymphoproliferative diseases such as Hodgkins lymphoma,non-Hodgkins lymphoma of low and intermediate grades, as well aspatients with acute lymphocytic leukemia (ALL), PLF levels are elevatedat early stages of disease and remain elevated as disease progresses.While the applicants are under no duty or obligation to explain themechanism of the invention, it may be that the infected lymphocytes inthe case of AIDS or the malignant lymphocytes in the case of lymphomaand leukemia are the cellular sources of the PLF which is expressed atelevated levels per cell during immunosuppression. Accordingly, in AIDSpatients, as the population of infected lymphocytes declines, the levelsof PLF detected will diminish. By contrast, in lymphoma and leukemia asthe population of malignant lymphocytes proliferate, the levels of PLFwill remain elevated.

The detection of PLF in patient samples may be accomplished by any of anumber of methods. A convenient approach for detecting PLF, as describedin more detail infra, involves immunoassays that utilize monoclonalantibodies which define and bind to PLF exclusive of isoforms of adultferritin. Such monoclonal antibodies may be used in any immunoassayformat for the detection of PLF, including but not limited to,enzyme-linked immunosorbant assays (ELISA), radioimmunoassays,fluorescent immunoassays, etc. in a "sandwich" or competition format orcytotoxic assay systems. In specific embodiments described herein, twotypes of monoclonal antibodies are described which are particularlyuseful in a sandwich immunoassay format: one monoclonal antibody crossreacts with both PLF and adult ferritin, whereas a second monoclonalantibody specific for PLF does not cross react with adult ferritin. Thecross-reactive antibody can be used in a sandwich immunoassay as a"capture antibody" to capture all isoforms of ferritin present in thesample, e.g., both PLF and adult ferritin. The PLF-specific monoclonalantibody can be labeled and used to detect captured PLF whereas thecross-reactive monoclonal antibody can be labeled and used to detect allcaptured isoforms of ferritin.

The subsections below describe the characterization of PLF, monoclonalantibodies that define PLF and the diagnostic and prognostic uses of PLFin autoimmune conditions. The invention is demonstrated by way ofexamples in which levels of serum PLF and adult ferritin in HIV infectedpatients as well as patients with lymphoma or leukemia and certainautoimmune diseases were monitored during the course of disease usingmonoclonal antibodies for PLF and adult ferritin in an ELISA sandwichformat.

5.1. PLACENTAL FERRITIN AND MONOCLONAL ANTIBODIES SPECIFIC FOR PLACENTALFERRITIN

Two monoclonal antibodies, CM-H9 which reacts exclusively with PLF, andCM-G8 which cross reacts with both PLF and adult ferritin, were used tocharacterize placental ferritin. Placental ferritin(s) reactive withCM-H9 was most acidic in comparison to CM-G8 reactive molecules,indicating structural heterogeneity in human placenta ferritin. A threesubunit structure of placenta ferritin was revealed by our analysis: an18 Kd light (L) subunit and 20 Kd heavy (H) subunit as well as a 43 Kdsubunit. The 18 Kd L and 20 Kd H subunits have been shown previously forspleen and liver ferritins; however, the third high molecular weightsubunit (43 Kd) seems to be unique for human placenta ferritin. CM-H9reactive placental ferritin was composed only of the 43 Kd subunit. This43 Kd subunit could not be further dissociated under exhaustive reducingconditions.

The 43 Kd unique subunit of human placenta ferritin so identifiedappears to be either part of the ferritin molecule or associated withit. This notion is based on the findings that this subunit contains theCM-G8 reactive antigenic epitopes present in spleen and liver ferritin.Furthermore, CM-H9 reative ferritin contained measurable amount of ironas was evident by its reactivity with potassium ferrocyanide and maytherefore be considered as a placenta associated isoferritin. UsingCM-H9 and CM-G8 to analyze fractions of PLF eluted from a DEAE columnrevealed that the 43 Kd subunit can occur as a homopolymer or as a dimerwith the L or H chain.

Some structural similarities between the 20 Kd H subunit and the 43 Kdsubunit were observed. Both subunits were sensitive to V8 proteolysisand both lost their antigenic reactivity with CM-G8 following SDStreatment under reducing conditions. It may be that the high molecularweight subunit (43 Kd) of placenta ferritin is either a stable dimer orprecursor of H subunit (20 Kd) (See also, Parhami-Seren and Moroz, 1986,G.I. Pat. Clin. 1(1):17).

Finally, the unique 43 Kd subunit present in placenta ferritin whichreacts with CM-H9 was also found in ferritin molecules synthesized bybreast cancer cells and not in ferritin synthesized by normal breastcells. Furthermore, CM-H9 reactive ferritin was detected in blood ofbreast cancer patients but not in healthy individuals. We thereforesuggest that the CM-H9 reactive 43 Kd subunit is characteristic ofcarcino fetal ferritin.

5.1.1. THE MOLECULAR HETEROGENEITY OF PLACENTA FERRITIN

Placenta ferritin obtained following DEAE-cellulose chromatography (asdescribed in Section 6.1 infra) was subjected to isoelectric focusing(IEF), and was further reacted With either ¹²⁵ I-CM-H9 or ¹²⁵ I-CM-G8McAb. ¹²⁵ I-CM-H9 McAb reacted with placenta ferritin at pH ranging from4.7-5.2. On the other hand, spleen ferritin isoelectrofocused on agar,did not react with ¹²⁵ I-CM-H9 McAb. ¹²⁵ I-CM-G8 McAb reacted withplacenta ferritin focused at pH 5.1-5.4, and with spleen ferritinfocused at pH of 5.4-5.5. The results of IEF indicate that placentalferritin is heterogeneous. The most acidic ferritin (pI 4.7-5.0),reacted with CM-H9 McAb whereas the less acidic molecules (pI 5.1-5.2)reacted with both CM-H9 and CM-G8 McAbs. It was also found that spleenferritin focused at pH 5.4-5.5 reacted with ¹²⁵ I-CM-G8 McAb whereas noreactivity was observed with ¹²⁵ I-CM-H9 McAb at such pH.

The PLF fractions eluted from the DEAE-cellulose column (as described inSection 6.1, infra) were further analyzed by ELISA assays using thefollowing capture/detection antibodies: (a) CM-H9 capture/CM-H9detection; (b) CM-G8 capture/CM-G8 detection; and (C) CM-G8capture/CM-H9 detection. Results shown in FIG. 7A and 7B demonstrate therelative quantities of the different PLFs in these fractions. Matzner etal., (1985, Brit. J. Haematol. 59:443-448) reported that Fraction IIdemonstrates the greatest biological activity (i.e., immunosuppressiveeffect on T cell function in vitro) whereas Fraction I, does notdemonstrate such activity. Interestingly, our results reveal that themore active Fraction II contains the greatest amount of CM-H9 reactivePLF.

5.1.2. THE SUBUNIT STRUCTURE OF PLACENTA FERRITIN REACTIVE WITH CM-H9AND CM-G8 MONOCLONAL ANTIBODIES

Separation of placenta ferritins by SDS-PAGE under reducing conditionsfollowed by immunoblotting with ¹²⁵ I-CM-H9 or ¹²⁵ I-CM-G8 revealed that¹²⁵ I-CM-H9 reacted with a single subunit structure (43 Kd) of placentalferritin. No reactivity of ¹²⁵ I-CM-H9 McAb was observed with spleenferritin. No reactivity with ¹²⁵ I-CM-G8 McAb could be observed withplacenta or spleen ferritin. These results suggest that the CM-G8reactive ferritin antigenic determinants of both placenta and spleenwere sensitive to SDS treatment under reducing conditions. Furtherimmunoblotting experiments were carried out with polyclonal rabbitanti-human spleen ferritin and ¹²⁵ I-protein A. A single band of 18 Kdwas evident in both placenta and spleen ferritin following SDS-PAGE.

Since CM-G8 reactive determinants of both placenta an spleen ferritinscould not be detected following SDS treatment, experiments were designedin which affinity purified placenta ferritin was radiolabeled andimmunoprecipitated with the monoclonal antibodies prior to SDS-PAGE. ¹²⁵I-CM-H9 reactive ferritin immunoprecipitated with differentconcentrations of CM-H9 McAb and electrophoresed on SDS-PAGE revealed asingle subunit structure of 43 Kd. This subunit structure was notfurther dissociated following exhaustive reducing conditions (boilingfor 10 minutes in 2% SDS and 5% β-mercaptoethanol or in 6M urea). On theother hand, ¹²⁵ I-CM-G8 reactive ferritin immunoprecipitated withdifferent concentrations of CM-G8 McAb and subjected to SDS-PAGE, underthe above described conditions exhibited three distinct subunits of 43,20 and 18 Kd. These results indicate the presence of a 43 Kd subunitstructure common to both CM-H9 and CM-G8 reactive ferritins, whereasonly the CM-G8 reactive ferritin contained, in addition to the 43 Kdsubunits, the H and L chains.

5.1.3. ENZYMATIC DIGESTION OF PLACENTA ISOFERRITIN WITH V8 PROTEASE

Further experiments were carried out to determine the sensitivity of theferritin subunits to limited proteolysis by V8 protease. Most of the 43Kd subunit of CM-H9 reactive ferritin was digested following a 60 minuteincubation with V8 protease. However, a complete digestion of thissubunit was not achieved even following incubation for 120 minutes. WhenCM-G8 reactive ferritin was treated with V8 protease, the 43 Kd as wellas the 20 Kd subunits were completely digested

5.2. DETECTION OF PLACENTAL FERRITIN

A convenient method for the detection of PLF in patient samples involvesimmunoassays that utilize monoclonal antibodies which define PLFexclusive of isoforms of adult ferritins. Such antibodies may beconfigured in a variety of immunoassays including, but not limited toELISA, radioimmunoassays, fluorescent immunoassays, etc. in a"sandwich", competition, or cytotoxic/target cell format.

In specific embodiments described herein, two types of monoclonalantibodies are described which are particularly useful for such assays:monoclonal antibodies such as CM-G8, which cross react with both PLF andadult ferritin, and monoclonal antibodies such as CM-H9, which arespecific for PLF and do not cross react with adult ferritin. Theseantibodies may be used in a number of configurations in a sandwich typeassay to monitor both PLF and adult ferritin levels in a patient. Forexample, a PLF specific antibody can be used as both the capture anddetection antibody to monitor levels of PLF. Alternatively, across-reactive antibody can be used to capture all isoforms of ferritin(i.e., both PLF and adult ferritin) in the sample; in this case, a PLFspecific antibody can be used to detect the PLF isoforms in the sample,and a cross- reactive antibody can be used on a duplicate sample todetect all cross reactive ferritins present in the sample. The resultsof such a sandwich assay can provide a profile of the relative levels ofPLF and adult ferritin in a patient sample.

In another embodiment of the invention, the PLF and cross-reactiveantibodies may be differentially labeled and used to determine therelative proportions of PLF and adult ferritin in a sample. In suchapplications, each antibody may be labeled with a different fluor,chromophore, photoemitter, or enzyme to produce a different fluorescentor colorimetric signal. The measurement of each signal could provide fora differential analysis of PLF and adult ferritin in a single sample.

The immunoassays of the invention are not limited to the use of CM-H9and CM-G8 monoclonal antibodies. In fact, other monoclonal antibodieswhich are functionally equivalent to CM-H9 and CM-G8 are contemplatedfor use in accordance with the invention. To this end, CM-H9 and CM-G8can be used to isolate the respective PLF and adult ferritin moleculesin order to produce functionally equivalent antibody molecules that canbe used in accordance with the invention. Such monoclonal antibodies canbe prepared using any techniques which provides for the production ofantibody molecules by continuous cell lines in culture. For example, thehybridoma technique originally developed by Kohler and Milstein (1980,Sci. Am. 243(4): 66-74) as well as other techniques which have morerecently become available, such as the human B-cell hybridoma technique(Kozbor et al., 1983, Immunology Today 4:72) and the EBV-hybridomatechnique to produce human monoclonal antibodies (Cole et al., 1985,Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96)and the like are within the scope of the present invention. Antibodymolecules produced by such methods which define the CM-H9 and CM-G8epitopes (e.g., those that competitively inhibit the binding of CM-H9 orCM-G8 to their target antigens) would be selected for use in accordancewith the invention.

The present invention, however, is not limited to the use of monoclonalantibody molecules for the detection of PLF. As more or differentmethods for the isolation and characterization of PLF are developed, itwill become apparent to the skilled artisan that such techniques couldalso be used in accordance with the invention to monitor patient samplesfor PLF.

5.3. PLACENTAL FERRITIN AND IMMUNOSUPPRESSIVE CONDITIONS

The results presented in the Examples infra are discussed below. Inparticular, the data presented in Section 7 demonstrate that PLF isproduced in HIV-infected patients, and that the presence of PLF couldplay a significant role in the pathogenesis of immunodeficiency in AIDS.The data presented in Sections 8 and 9 demonstrate that PLF levels areelevated in patients with certain lymphomas and leukemias and autoimmunediseases that are characterized by immunosuppression.

The availability of a distinct monoclonal antibody which specificallydefines PLF, enabled us to design an assay to measure serum levelsspecifically of PLF, independently of the amount of adult ferritin. Fromthese measurements, isoferritin profiles were derived which may serve asprognostic indicators for the progression of HIV seropositive patientsto ARC and to AIDS.

5.3.1. PLACENTAL ISOFERRITIN IN HIV INFECTION

PLF was measured in patients classified by the following stages: StageA, HIV seropositive but no clinical manifestations or physical findings;Stage B, lymphadenopathy and/or splenomegaly; Stage C, clinical symptomsor findings related to ARC; Stage D, Kaposi's sarcoma, lymphoma, or CNS(central nervous system) disease without systemic opportunisticinfections; and Stage E, opportunistic infections originally defined bythe CDC as diagnostic of AIDS. The results reveal that the majority ofHIV infected patients with early clinical manifestations (Stage B),exhibit significant elevations in the concentration of serum PLF. Theseelevations are maintained in most patients at Stage C (ARC). Incontrast, further progression of the disease into AIDS is accompanied bya significant elevation in the level of total serum ferritin, but by adiminution in PLF concentration. The increase in normal ferritin levelsin AIDS patients has been previously reported by other investigators(Blumberg, B., et al., 1984, Lancet 1:347; Gupta, S., et al., 1986, J.Clin. Lab. Immunol. 20:11-13).

Although the Applicants are under no duty to explain the invention, wesuggest that in HIV infected individuals, the increase in serum PLF isclosely associated with the development of lymphadenopathy and laterclinical manifestations of AIDS-related complex. Increases in the levelof normal ferritin appear to be associated predominantly with theprogression of the disease from ARC to AIDS. Subjects with clinicallylatent HIV infection (stage A) do not show rises in serum isoferritins.

The cellular origin of the serum PLF in HIV infected patients is not yetknown. However, the observation that with the progression of thedisease, decreases in the serum level of PLF positively correlated withdecreases in the total number of CD4⁺ lymphocytes, suggests that PLF mayoriginate from these CD4⁺ cells. Indeed, both serum PLF levels and thetotal number of CD4 lymphocytes decrease during disease progression.However, the proportion of HIV infected CD4⁺ lymphocytes within thediminishing population of these cells probably increases during thistime. This increase in the proportion of HIV infected CD4⁺ lymphocytesmay explain the observed increase in the ratio of serum PLFconcentration per individual CD4⁺ lymphocyte in stage E (FIG. 2). Theseresults suggest that serum PLF originates from the HIV infected CD4⁺cells, and that therefore, the ratio of serum PLF levels per CD4⁺lymphocyte may be used as a diagnostic indicator of the degree ofinfection. In sum, while the highest absolute concentrations of serumPLF were associated with early stage HIV infection, the ratio of theconcentration of PLF per circulating CD4⁺ lymphocyte may be useful as anindex of the degree of cellular infection.

The results also demonstrated that in HIV infected subjects, thereexists a subset of CD8⁺ cells (15.2±6.4%) to which PLF is bound, maskingthe CD8 antigen. It is unclear whether the receptor for PLF is the CD8antigen, or a site close to it such that CD8 is masked by PLF via sterichindrance. Since the majority of CD8⁺ lymphocytes were neitherPLF-positive nor blocked in their reaction with McAb-T8, the formerpossibility seems very unlikely. The masking of a T cell surfacereceptor by isoferritin derived from tumors has been observed in cancerpatients (Hann, H.-W. L., et al., 1984, Nature (London) 265:755-756;Moroz, C., et al., 1977, Clin. Exp. Immunol. 29:30-35; Moroz, C., etal., 1977, N. Engl. J. Med. 296:1175; Moroz, C., et al., 1977, CancerImmunol. Immunother. 3:101-104; Moroz, C., et al., 1984, Cancer54:84-89). In these patients, isoferritin inhibited E-rosette formationby the masked T cells. In AIDS patients, the E-receptor (T11 antigen)was not masked when tested with anti-T11 McAb. The discrepancy betweenthe above observations may lie in the ligands used to identify theE-receptor.

It is perhaps significant that the surface PLF was removed by treatmentof HIV infected lymphocytes with levamisole, but not by parallelincubation in complete tissue culture medium. The incubation withlevamisole resulted in unmasking of the normal CD8 surface marker. Thisobservation is compatible with previous findings on the unblockingeffect of levamisole on lymphocytes from patients with Hodgkin's diseaseand breast cancer (Moroz, C., et al., 1977, Cancer Immunol. Immunother.3:101-104; Ramot, B., et al., 1976, N. Engl. J. Med. 294:809).Levamisole has been shown to act as an immunopotentiating drug (Levo,Y., et al., 1975, Biomedicine 23:198-200; Nekam, K., et al., 1981,Immunopharm. 3:31-40) yet its mode of action is not yet understood.

The pattern of isoferritin expression across the clinical spectrum ofHIV infection suggests that PLF may play a role in the pathogenesis ofprogressive immunodeficiency, as it appears to do in the pathogenesis ofHodgkin's disease. A small proportion of peripheral blood lymphocytes ofnormal subjects (up to 6-7%) both in the present study and in prioranalyses (Moroz, C., et al., 1984, Cancer 54:84-89), most likely withinthe CD8 pool, binds PLF. This population appears to be expanded in HIVinfected patients. In addition, serum PLF rises dramatically inrelatively early HIV infection, corresponding to the period of maximallymphoid activation in clinical stages B and C. Preliminary data fromWalker et al. (1986, Science 234:1563-1566) suggests that certain CD8lymphocytes can inhibit HIV proliferation in vitro. Hypothetically, PLFcould inhibit the immunocompetence of these CD8 cells, therebycontributing to the progressive expression of HIV that is characteristicof late-stage disease (id.).

An explanation for the rise of PLF early in HIV infection, followed by adecrease later in infection, is so far lacking. One possibility,supported by our preliminary data, is that trans-activating viral gene(tat III) products increase PLF mRNA expression in virus-infected CD4cells. The depletion in the total number of these cells late in AIDScould explain the observed declines in serum PLF concentration, duringwhich time total ferritins increase in response to nonspecific stimuli(e.g., secondary infection) as acute-phase reactants.

The finding that levamisole, a known immunopotentiator, enhances theelution of PLF from a CD8⁺ subset might indicate a role for this drug inthe therapy of HIV infections, particularly if used in early stages ofthe disease.

6. PREPARATION OF MONOCLONAL ANTIBODIES SPECIFIC FOR PLACENTAL FERRITIN

The subsections below describe the preparation of placental ferritin andmonoclonal antibodies (e.g., CM-H9 McAb) that define a unique epitope ofplacental ferritin (PLF). These antibodies do not cross react withspleen or liver ferritin.

6.1. PREPARATION OF PLACENTAL FERRITIN

Placental ferritin was prepared from human placenta by a modification ofthe method used by Beamish et al. (1971, J. Clin. Path. 24:581).Placental tissue (500 g) was sliced and water added to a total volume of2000 ml. After homogenization the tissue suspension was heated to 75° C.for 20 minutes. The supernatant, after cooling and centrifugation at10,000 rpm for 15 minutes, was treated with acetic acid to bring the pHto 4.6. The precipitated protein was removed by centrifugation at 10,000rpm for 15 minutes and a clear supernatant was adjusted to neutral pHwith dilute NaOH. When the clear brown supernatant was ultracentrifugedat 100,000×g for 240 minutes the suspended ferritin collected in a smallbutton at the bottom of the tube. The precipitate was redissoved in 0.9%saline and further purified by passage through a Sephadex G200 column.The ferritin fraction from this column was passed through a DEAEcellulose anion exchange resin using Tris-HCl buffer at pH 7.5 and a0.02-0.5M gradient. Three protein peaks were obtained, the most acidicpeak pI=4.8 was collected and used for analysis. Its purity was shown byisoelectric focusing and immunoelectrophoresis against anti-ferritinserum and anti-human whole serum. This protein was used for theimmunization of mice as described below.

6.2. PREPARATION OF MONOCLONAL ANTIBODIES THAT BIND TO PLACENTALFERRITIN

The following protocol was used to produce monoclonal antibodies thatbind to PLF but which may also cross react with other isoferritins. Theprotocol and monoclonal antibodies made are described in the followingapplications: copending application Ser. No. 07/148,133 now U.S. Pat.No. 4,882,270, filed Jan. 22, 1988, issued Nov. 21, 1989 a continuationof application Ser. No. 568,275 filed Jan. 4, 1984, a continuation inpart of application Ser. No. 373,715, filed Apr. 30, 1982 which claimspriority to Israel application Serial. No. 62879, filed May 15, 1981,each of which is incorporated by reference herein in its entirety. Seealso, Moroz et al., 1985, Clinica Chemica Acta 148:111-118.

6.2.1. MATERIALS AND METHODS

The following media and solutions were used in the preparation of themonoclonal antibodies:

a. RPMI-O (No FCS)

b. RPMI 1640-HY

500 ml sterile distilled water

55 ml 10 x RPMI-1640

6 ml 1.0N Sodium Hydroxide

14 ml 7.5% Sodium Bicarbonate

6 ml Pen/strep)

10 ml Glutamine)+DMEM

86.5 ml FCS)

c. RPMI-HY-HATD-day 0 to day 7

For 100 ml of medium

95 ml RPMI -1640+20% FCS

1.0 ml Pyruvate (100x)

2.0 ml 50×HAT

2.0 ml 50×deoxycytidine

d. RPMI - HY - HT- day 8 to day 14

For 100 ml of medium 97 ml RPMI-1640+20% FCS 2.0 ml 50 x HT 1.0 mlPyruvate (100x)

For Hybrids from day 15/onwards use RPMI-1640+20% FCS and pyruvate, ormaintain in RPMI-HY-HT.

e. PEG 33 and 25% w/v

Must be ordorless and white. For 100 ml autoclave relevant wt in gramsin a glass bottle at 15 lbs for 10-15 minutes. When bottle is coolenough to hand hold (about 50° C.) add RPMI 1640-0 to make up to 100 ml,swirl to mix, store at room temperature.

f. HATD - Final concentrations of reagents

H=Hypoxanthine 10⁻⁴ M

A=Aminopterin 10⁻⁶ M

T=Thymidine 2×10⁻⁵ M

D=Deoxycytidine 2×10⁻⁶ M

HT Stock 100x-100 cc

Thymidine (M.W. 242.33): 0.04846 g

Hypoxanthine (M.W. 136.1): 0.1361 g.

Add H₂ O up to 100 ml and warm to 60°-70° C. to dissolve. Readjust finalvolume with double distilled water (dd H₂ O). Dilute to 50x, sterilefilter (0.2μ) and store 2 ml aliquots at -20° C.

g. A Stock 1000 x -100 cc

Aminopterin (F. W. 440.4): 0.44 g

Bring to 50 ml with dd H₂ O, add 0.1N NaOH dropwise until aminopterindissolves. Bring final volume to 100 ml with dd H₂ O. Adjust volume to100 ml. Sterile filter (0.2μ) and store at -20° C.

h. D Stock 100 x -100 cc

Deoxycytidine (M.W. 227.2): 0.00454 g

Dissolve in dd H₂ O, adjust to 100 cc, dilute to 50x stock. Sterilefilter (0.2μ) and store at -20° C.

i. HAT - 50x - 200 ml

Combine 100 ml 100×HT with 10 ml 1000×A+90 ml dd H₂ O=50×HAT. Sterilefilter (0.2μ) and store 2 ml aliquots at -20° C.

6.2.2. IMMUNIZATION PROTOCOL

Balb/c female mice (4-6 weeks old initially) were immunized with 3weekly inoculations of 50 μg acidic placental ferritin (prepared asdescribed in Section 6.1, supra) in complete Fruends adjuvant.Hybridizations were performed 3 days after the last injection of 10 μgacidic placental ferritin. Hyperimmune mice were rested at least onemonth before the final boost.

6.2.3. SPLEEN CELL-MYELOMA FUSIONS

Spleen cells were prepared as follows:

a. Spleens were removed from mice in RPMI-O;

b. Rinsed 2x in petri dish with RPMI-O;

c. Teased apart in RPMI-O with 18 ga. needles;

d. Cell suspension transferred to a tube and large chunks of tissuesettled out;

e. Single cell suspension removed to a new tube spun at 800 RPM (160×g)5 min; Red blood cells lysed with 0.83% NH₄ Cl, pH 7.5;

f. Cells washed 3x with RPMI-O, resuspended in same;

g. Cells counted with Trypan Blue.

Myeloma cells used for fusion, PB/NS1/1-Ag4-1 were grown in RPMI-1640with 20% Fetal Calf Serum (FCS) and prepared as follows:

a. Myeloma cells were removed from culture flasks with gentle pipettinginto 50 ml Falcon/Corning tube;

b. Spun down at 900 RPM (200×g) 5 minutes;

c. Washed 1x with RPMI-O, resuspended in same and counted with TrypanBlue.

Spleen cells were fused to the myeloma cells as

a. Spleen and myeloma cells were combined in a 10:1 ratio in a single 50ml conical Falcon/Corning disposable centrifuge tube;

b. Cells were pelleted at 900 RPM (200 x g) for 5 minutes;

c. Medium was aspirated as completely as possible;

d. All solutions and media used from now on were at room temperature;centrifuge tube with cell pellet was immersed in a bath at 37° C., andthe following was added accompanied by gentle stirring: 0.2 ml 33% PEG1500 for 1 minute, centrifuged at 200×g for 5 minutes. Cells wereresuspended and stirred gently for 1 minute followed by the addition of5 ml RPMI-O gentle stirring and additon of 5 ml RPMI-O 20% Fetal CalfSerum. Hybrid mixture looked like a poorly resuspended cell suspensionat this point with many small clumps;

e. The mixture was pelleted at 200×g 5 minutes;

f. Cells were resuspended in RPMI-HY-HATD (at 37° C.) at a concentrationof 3×10⁶ /cc by squirting medium onto the cell pellet;

g. Hybrids were plated out in flat bottom 96 well plates by adding 2drops of cell suspension from a 5 ml pipet or with multi-pipettor usingcut off tips (about 65 microliters), containing 100-120 RPMI-HY-HATD(approx. 2×10⁵ cells);

h. Control wells containing NS-1 cells+RPMI-HY-HATD at 1×10⁶ /ml wereset up;

i. Plates were cultured for 7 days;

j. On Day 8 and twice a week thereafter, half of the culture medium wasremoved by careful aspiration and fed with 80-100 microliters ofRPMI-HY-HT medium;

k. Positive wells were screened for at 3 and 4 weeks afterhybridization.

6.2.4. SCREENING PROTOCOLS

Screening and determination of the specificity of the monoclonalantibodies was performed by a hemagglutination test. Embryonic placentaand adult spleen ferritin were coupled to Ox red blood cells Ox RBC byCrCl₂. 50 μl of increasing dilutions (starting at 1:10 of hybridomaculture medium supernatant were mixed with 10 μl of adult of embryonicferritin Ox RBC and hemagglutination determined.

Supernatants of clones giving a hemagglutination titer of at least1:1000 were selected.

A clone, designated CM-OF-3 was selected (hereinafter referred to asCM-3). The clone CM-3 produces a monoclonal antibody which is specificfor embryonic ferritin and it cross-reacts with both adult and embryonicferritin.

Another clone, designated CM-G8, produces a monoclonal antibody whichbinds to placental ferritin and cross reacts with spleen and liverferritin. CM-G8 defines the same epitope recognized by CM-3.

6.3. PREPARATION OF MONOCLONAL ANTIBODIES SPECIFIC FOR PLACENTALFERRITIN AND NOT CROSS REACTIVE WITH NORMAL FERRITIN

The following protocol was used to prepare monoclonal antibodies thatwere specific for PLF and which did not cross react with otherisoferritins. In particular, the monoclonal antibody, CM-3 describedabove was used to block the cross-reactive determinants of fetal andadult ferritin, in order to produce a different monoclonal antibody,CM-H9, which is directed to a specific fetal determinant.

6.3.1. IMMUNIZATION AND FUSION PROTOCOLS

The following immunization and fusion procedure was used to obtainmonoclonal antibodies that define a unique epitope of PLF and do notcross react with other isoferritins. Embryonic ferritin isolated fromhuman placenta (the protein of pI 4.8 isolated as described in Section6.1 supra) was reacted with monoclonal antibodies CM-3 in the followingratio: embryonic ferritin (90 μg in PBS) was mixed with Ascites fluidfrom BALB/c mouse containing CM-3 antiferritin monoclonal antibodies (10mg/ml).

The mixture was incubated for 30 minutes at 37° C. followed by overnightincubation at 4° C. The mixture was centrifuged at 10,000×g, theprecipitate formed was discarded, and the supernatant was used forimmunization. Each BALB/c mouse was immunized with the above supernatant

with complete Fruend's adjuvant, injected intradermally once a week for3 weeks. A booster immunization of one fifth of the above dose wasinjected intraperitoneally.

After 3 days from boost, mouse spleen was aseptically removed and fusionwas performed by incubating spleen cells with 10⁷ /P3-NSI/1-Ag4 myelomacells as set out above. Positive clones were identified as describedabove. A clone designated as CM-OF-H9 (hereinafter referred to as CM-H9)was obtained.

6.3.2. CHARACTERIZATION OF MONOCLONAL ANTIBODY CM-H9

The CM-H9 monoclonal antibody belongs to the IgG class; it does not formprecipitates with ferritin, it binds rabbit complement. In the asciticfluid obtained, the antibody content was about 7 mg per ml. One ml ofascitic fluid binds about 2 mg of placental ferritin and none of adultspleen or liver ferritin.

6.4. IMMUNOASSAYS FOR LYMPHOCYTE BOUND PLACENTAL FERRITIN USING CM-H9MONOCLONAL ANTIBODY

Monoclonal antibody, CM-H9, can be used in immunoassays for thedetection of PLF in test samples such as serum or PLF bound tocirculating peripheral blood lymphocytes (PBL). The presence of PLF canbe determined by using CM-H9 in any type of immunoassay system,including but not limited to ELISA, radioimmunoassay or cytotoxic assays(where cellular targets are involved).

In general, the assay for the detection of placental ferritin which isbound to peripheral blood lymphocytes may be carried out by (a)isolating lymphocytes from peripheral blood, and (b) determining thepresence of PLF on the lymphocytes using a conventional type of assaybased on the use of the novel monoclonal antibodies, specific for PLF.

According to a perferred embodiment the test may be carried out asfollows:

a. Lymphocytes are isolated from peripheral blood by Ficoll-Hypaquegradient centrifugation;

b. The presence or absence of PLF bound to the surface of thelymphocytes is determined by any conventional type of assay, such asELISA, cytotoxic test or radioimmunoassay.

6.4.1. COLLECTION OF LYMPHOCYTES

Lymphocytes are collected as follows:

(a) Collect 15 ml blood into a heparin-containing blood collection tube;dilute 1:2 in PBS pH 7.2

(b) Underlay the cell suspension with 10 ml Ficoll-Hypaque densitysolution (1.077 gm/ml).

(c) Centrifuge for 30 minutes at 300×g at room temperature.

(d) Collect mononuclear cells from the medium:Ficoll-Hypaque interfaceusing a Pasteur pipette and transfer to a new 15 ml tube.

(e) Wash cells 3 times by suspension in 15 ml wash medium (PBS, pH 7.2)and centrifuge at 300×g for 10 minutes at 4° C.

(f) Resuspend in wash medium and determine cell number.

6.4.2. RADIOIMMUNOASSAY PROCEDURES

Two procedures were followed for radioimmunoassay:

A. Radio-Immuno Assay - 1

Peripheral blood mononuclear cells were isolated by Ficoll-Hypaquegradient centrifugation. The test was performed in triplicate (A Blank;B Test sample):

1. Dispense 2×10⁶ to 3×10⁶ cells into each of six test tubes; pelletcells by centrifugation at 300×g for 10 minutes.

2. Add NRS (normal rabbit serum) 20 μl diluted 1:10 in PBS, incubate 60minutes at 4° C.

3. Add 30 μl of ascites fluids (dilution 10⁻⁵ in 5% BSA) to each of 3tubes.

A. Control ascites fluid containing an IgG non-specific monoclonalantibody, non-reactive with PLF.

B. CM-H9 monoclonal antibodies. Mix well and incubate at roomtemperature for 2 hours.

4. Wash cells twice with 10 ml RPMI-1640 by centrifugation at 300×g for10 minutes at 4° C.

5. Add 0.1 μCi of I¹²⁵ rabbit anti-mouse IgG (¹²⁵ -I Rabbit IgG 1μCi/μg) incubate 60 minutes at 4° C., wash twice with cold RPMI-1640 asin 4, count radioactivity.

Positive test:

Cpm A- CpmB<500; or

Cpm A:Cpm B>1.6.

B. Radio Immuno Assay - 2

After Stage 1, RIA - 1, the test procedure is continued as follows:CM-H9 F(ab)₂ is obtained by peptic digestion of CM-H9 IgG according toUtsumi and Karush (1965, Biochem. 4:1766). Control F(ab)₂ is similarlyobtained from the non-specific IgG (see control of RIA-1). The F(ab)₂fragments thus obtained are used as follows:

Tube A: Control F(ab)₂ in 5% BSA in PBS (pH 7.2) 0.025% sodium azide.

Tube B: CM-H9 F(ab)₂ in 5% BSA in PBS (pH 7.2) 0.025% sodium azide.

Incubate for 60 minutes at room temperature, wash once with 2 ml of 1%BSA in PBS (pH 7.2); add ¹²⁵ I-labeled ligand to test tubes A and B(about 10⁵ cpm); either ¹²⁵ I-labeled PLF or a complex of ¹²⁵I-polyclonal anti-PLF with PLF. The complex is preformed atantigen/antibody molar ratios of 1:1 or up to 1:2, pre-incubated witheach other at room temperature for 1 hour. Incubate the labeled ligandtogether with cells for 1 hour at room temperature, wash twice with 1%BSA in PBS (pH 7.2) to remove unbound labeled ligand and count. If Bexceeds A the test is positive.

6.4.3. CYTOTOXIC ASSAY PROCEDURE

Test is performed in duplicates: (A) Control; and (B) Test Sample.

a. Suspend PBL at a density of 5×10⁶ cells/ml in RPMI-1640

b. Place 150 μl of PBL into each of four 12×75 mm test tubes. Addascites fluids (30 μl dilution 10⁻⁴): A. Control ascites fluid (2tubes); B. CM-H9 (2 tubes). Incubate 45 minutes at 4° C.

c. Add rabbit complement (100 μl) diluted 1:5 in PBS) and incubate 60minutes at 37° C. with slow agitation.

d. Count viable cells with Trypan blue.

Positive Test: ##EQU1##

6.4.4. RESULTS: REACTIVITY OF CM-H9 MONOCLONAL ANTIBODY WITH LYMPHOCYTESIN CERTAIN DISEASES

Using the assays described above, the two monoclonal antibodies CM-H9and CM-3 were used to screen serum and PBL obtained from patients withvarious diseases as well as disease-free subjects. The results presentedin Table I below indicate that the two antibodies make possible rapidand convenient detection of malignancies of the breast and of Hodgkin'sdisease, and provide for differentiation of these from thalassaemia,which results in an increase of normal ferritin.

                  TABLE I                                                         ______________________________________                                        REACTIVITY OF PLF MONOCLONAL                                                  ANTIBODY WITH DIFFERENT PATIENT SAMPLES                                       Source of Human   Anti-PLF  Anti-Ferritin                                     Ferritin          CM-H9     CM-3                                              ______________________________________                                        1.    Adult spleen    -         +                                                   (Thalassaemia)                                                          2.    Normal serum    -         +                                             3.    Breast cancer (PBL)                                                                           +         +                                             4.    Breast cancer (serum)                                                                         +         +                                             5.    Hodkin's        +         +                                                   Disease (spleen)                                                        6.    Benign breast   -         -                                                   disease (PBL)                                                           7.    Benign breast   -         +                                                   disease-serum                                                           ______________________________________                                    

7. ISOFERRITINS IN HIV INFECTION: RELATION TO CLINICAL STAGE, CD8⁺LYMPHOCYTE BINDING AND THE PATHOGENESIS OF AIDS

In the examples detailed infra, placental isoferritins (PLF) were foundto be increased in sera of subjects infected with human immunodeficiencyvirus (HIV). PLF was quantified by use of a "sandwich" antigen captureELISA employing two monoclonal antibodies. Individuals withlymphadenopathy, with or without symptoms suggestive of AIDS-relatedcomplex, had the highest serum levels, which declined with progressiveimmunodeficiency. Total (normal) ferritins, in contrast, increasedprogressively with stage of disease. PLF was found on the cell surfaceof a subset of CD8⁺ lymphocytes and appeared to block detection of theCD8 antigen by specific monoclonal antibodies. Elution of PLF from thecell surface, achieved by incubation with levamisole but not by culturemedium alone, led to the unblocking of the CD8 determinant on thesecells. Profiles of isoferritins in HIV infection may thus provide cluesto prognosis. PLF, a physiologic down-regulator of hematopoiesis andcellular immunity, may be abnormally expressed via trans-activation byHIV gene products, and could play a role in the progressive immunedeficiency, marrow suppression and HIV expression that lead to AIDS.

7.1. MATERIALS AND METHODS 7.1.1. SUBJECTS

Sera from HIV seropositive patients were derived from material stored at-70° C. obtained during a previous study (Siegal, F. P., et al., 1986,J. Clin. Invest. 78:115-123), and from patients in an ongoing study.Patients were classified according to clinical stage with themodification that all subjects included were confirmed to be HIVseropositive. The stages were defined as follows: Stage A: HIVseropositive but without clinical manifestations or physical findings;B: lymphadenopathy and/or splenomegaly; C: clinical symptoms or findingsrelated to ARC; D: Kaposi sarcoma, lymphoma, or CNS (central nervoussystem) disease but without systemic opportunistic infections; E:opportunistic infections defining AIDS by original Center for DiseaseControl (CDC) criteria (Center for Disease Control, Update on acquiredimmune deficiency syndrome (AIDS)-United States, 1982, Morbid. Mortal.Weekly Rep. 31:507-514). Sera were also Obtained from 40 hematologicallynormal blood bank donors.

7.1.2. ISOLATION OF LYMPHOCYTES

Peripheral blood mononuclear cells were isolated from fresh heparinizedblood by Ficoll-Hypaque gradient density centrifugation.

7.1.3. MONOCLONAL ANTIBODIES

Monoclonal antibodies (McAb) T4, T8 and T11, reactive with CD4⁺, CD8⁺,and CD2⁺ cells, respectively, directly conjugated to fluorescein orphycoerythrin, were obtained from Coulter Immunology (Hialeah, Fla.).CM-H9 McAb which defines human placental ferritin and has been shown toreact exclusively with placental isoferritin and not with liver orspleen ferritins (Moroz, C., et al., 1985, Clin. Chim. Acta148:111-118). CM-G8 McAb was produced against human placentalisoferritin, but also reacts with human liver and spleen ferritins inaddition to PLF (id.).

7.1.4. FLOW CYTOMETRY AND IMMUNOFLUORESCENCE STAINING

CD4⁺, CD8⁺, and CD2⁺ cells were assayed by flow

CD4 cytometry using a Coulter Epics 5 cell sorter modified for two colorimmunofluorescence. The lymphocytes were directly reacted withphycoerythrin conjugated T4 McAb, and with fluorescein-isothiocyanateconjugated T8 McAb or T11 McAb, according to the manufacturer'sspecifications.

7.1.5. IMMUNOFLUORESCENCE STAINING OF ISOFERRITIN ON LYMPHOCYTEMEMBRANES USING CM-H9 McAb

Lymphocytes were washed twice at room temperature with phosphatebuffered saline (PBS), pH 7.2, containing 2% bovine serum albumin (BSA)and 0.01% sodium azide (PBS-BSA). Two aliquots, containing 1×10⁶mononuclear cells each, were incubated in 25 ul of diluted CM-H-9 McAbovernight at 4° C. A third aliquot, containing 1×10⁶ cells, wasincubated with 25 ul of mouse IgG (Coulter) as a negative control. Afterincubation, the mononuclear cells were washed three times in PBS-BSA,incubated with 25 ul fluorescein-conjugated F(ab')₂ fragments of goatanti-mouse IgG F(ab')₂ (diluted 1:2) (Capell) for 30 minutes at 4° C.,and washed again three times in PBS-BSA.

After centrifugation, the washed cell pellets were suspended in 20 ul ofPBS-BSA and examined on a microscope slide with a Leitz Orthoplanepifluorescence microscope, with an excitation wave length of 288 nm forquantitation of the CM-H9 McAb membrane stained lymphocytes. At least400 lymphocytes were counted. Monocytes were morphologically identifiedby their large size and abundant granular cytoplasm, and were excludedfrom the count.

In some experiments, double staining of membrane isoferritin and CD4antigen was carried out. Following the addition of CM-H9 McAb andFITC-anti-mouse F(ab')₂ IgG, the lymphocytes were washed twice withPBS-BSA and further incubated with phycoerythrin-anti-CD4 McAb (5 μl,Coulter) for 30 minutes at 4° C. The cells were washed twice with PBSand analyzed with a fluorescent microscope as described supra.

7.1.6. IMMUNOFLUORESCENCE STAINING OF ISOFERRITIN IN LYMPHOCYTECYTOPLASM USING CM-H9 McAb

Lymphocytes (1×10⁶) were centrifuged onto precleaned microscope slides(Cytospin, Shandon Scientific), air dried for 5 minutes, and fixed inabsolute methanol for 10 minutes at -15° C. Cells were washed once inPBS for 5 minutes and incubated with CM-H9 McAb (50 μl) overnight in amoist chamber at room temperature. After the incubation, the slides werewashed three times in PBS (5 minutes each) and further incubated withFITC-goat-anti-mouse F(ab')₂ (25 μl, Capell) for 30 minutes at roomtemperature. The cells were washed three times with PBS and examined forcytoplasmic fluorescence. Approximately 1-4×10³ cells were counted perslide.

7.1.7. LEVAMISOLE TREATMENT OF MONONUCLEAR CELLS

Levamisole (Sigma, St. Louis, Mo.) was added to whole blood or toisolated mononuclear cells to a final concentration of 40 μg/ml followedby incubation for 30 minutes at 37° C. as described by Ramot et al.(1976, N. Engl. J. Med. 294:809). The levamisole treated cells were thenmixed with the different monoclonal antibodies, in preparation for flowcytometry and immunofluorescent staining as described above.

7.1.8. QUANTITATIVE DETERMINATION OF SERUM ISOFERRITIN

Ferritin and placental isoferritin (PLF) were measured in the sera of161 HIV infected patients and in the sera of 40 blood bank donors usinga specific McAb ELISA as previously described (Moroz, C., et al., 1987,Exp. Hematol. 15:258-262). The "sandwich" ELISA method of Engval andPerlman (1972, J. Immunol. 109:129-132) as modified by Voller et al.(1975, Lancet 1:426), was used to develop the assays for ferritin andPLF.

In both the assay systems for ferritin and for PLF, the McAb CM-G8,which binds all isoferritins, was coupled to the solid phase as capturereagent. As previously shown, high concentration of normal ferritin didnot compete with the binding of PLF to the solid phase (Moroz, C., etal., 1987, Exp. Hematol. 15:258-262). For detection of the capturedantigen, alkaline phospatase-conjugated CM-G8 McAb was used formeasurement of ferritin, and enzyme-conjugated CM-H9 McAb was used formeasurement of PLF. The amount of placental ferritin that binds 2.5 pgof alkaline-phosphatase (AP)-conjugated CM-H9 McAb is defined as 10units of PLF (10 U PLF).

7.1.9. STATISTICAL ANALYSES

Data were analyzed with the statistical package EPISTAT, run on anIBM-AT personal computer, using Student's t-test, correlationcoefficient, linear regression and Chi-square.

7.2. RESULTS 7.2.1. SERUM LEVELS OF FERRITIN AND PLF IN PATIENTS WITHHIV INFECTION

The use of an ELISA specific for PLF enabled the measurement of PLFconcentration, independently of the amount of total ferritin, in theserum of HIV infected patients and healthy blood bank donors. Theresults of such measurements are shown in FIG. 1A and Table II.

                                      TABLE II                                    __________________________________________________________________________    RELATIONSHIP OF PLF AND FERRITIN TO ARC AND AIDS                                       Number of Subjects   Number of Subjects                                       with Serum PLF Levels Indicated                                                                    with Serum Ferritin Levels Indicated            SUBJECTS <10 U/ml                                                                            >10 U/ml                                                                            X.sup.2 *                                                                        P     <200 ng/ml                                                                           >200 ng/ml                                                                           X.sup.2 *                                                                        P                              __________________________________________________________________________    Normal Control:                                                                        36     4             37     3                                        HIV Infected                                                                  A        16     8    3.24                                                                             0.04  18     4      0.72                                                                             0.39                           B        19    30    22.36                                                                            2.3 × 10.sup.-6                                                               51     2      0.1                                                                              0.7                            C         8    17    21.09                                                                            4.4 × 10.sup.-6                                                               18     6      2.49                                                                             0.11                           D        26    11    3.6                                                                              0.058 14     23     23.29                                                                            1.49 × 10.sup.-6         E        33    18    6.51                                                                             0.011 17     37     32.55                                                                            <10.sup.-8                     __________________________________________________________________________     *Compared to normal control.                                             

As shown in FIG. 1A, the mean concentration of PLF was 10±31.5 U/ml inhealthy donors. It is noteworthy that 70% of the sera tested containedno detectable PLF, with only 10% having concentrations higher than 10U/ml (FIG. 1A, Table I). The most elevated concentrations of PLF(25±25.3 and 18.2±16 U/ml), significantly higher than normal (p<0.01),were observed in patients at early stages of clinically manifesteddisease (stages B, C) (FIG. 1A). Also, in contrast to the resultsobtained with normal sera, 61-68% of these patients, sera contained morethan 10 U/ml of PLF in their serum (FIG. 1A, Table II).

On the other hand, patients with more advanced HIV infection (stages D,E) had relatively lower mean serum levels of PLF (7.8±11.7, 9.7±14.7),which were not significantly different than those of normal controls(FIG. 1A). Furthermore, only 29.7% and 35.3% of the patients with AIDShad more than 10 U/ml of serum PLF (FIG. 1A, Table II).

In contrast to the above results, the normal ferritin levels roseprogressively as the disease progressed (FIG. 1B, Table II). Sixty-twoand 68.5% of those with advanced disease (stages D, E) had more than 200ng/ml of serum ferritin (FIG. 1B, Table II). Among HIV seropositivepatients without clinical or physical signs (stage A), the mean level ofPLF was slightly increased above normal to 20.7±34.2 U/ml, with 33% ofthe patients having more than 10 U/ml (FIG. 1A, Table II), which was notsignificantly different from that of normal controls. The total ferritinlevel was also not significantly different from that of healthy donors(FIG. 1B, Table II).

7.2.2. RELATIONSHIP OF HIGH SERUM PLF AND NORMAL FERRITIN TO DISEASEPROGRESSION

Contingency table analysis of the individual results exhibited in FIG. 1was carried out using a cutoff level of 10 U/ml for PLF (chosen because90% of normal control levels were below this value) and 200 ng/ml oftotal ferritin (chosen because 92.5% of normal control levels were belowthis value). The results obtained revealed a statistically significantrelationship between elevation of serum PLF level and the presence ofrelatively early stages of HIV infection (for stages B and C, p=23×10⁻⁶,and p=4.4×10⁻⁶, respectively), whereas an elevation in the totalferritin level was highly associated with advanced disease (for stages Dand E, p=1.49×10⁻⁶, and p<10⁻⁸, respectively) (Table II).

Correlations carried out between the number of CD4⁺ and CD8⁺ cells inthe patients' peripheral blood and serum levels of PLF and totalferritin, revealed a positive relationship (correlation coefficient of0.17, p<0.02) between the number of circulating CD4⁺ cells and the levelof serum PLF. PLF levels decreased with decreasing numbers ofcirculating CD4⁺ cells. It is noteworthy that the majority of patientswith the most advanced disease (stages D, E) which had undetectable PLF,had very low or undetectable CD4⁺ cells.

We next determined the ratio of the serum PLF level (in those withdetectable amounts) to CD4⁺ lymphocyte count (FIG. 2). Interestingly,the AIDS patients (stage E) had a significantly higher ratio of PLF(U/CD4⁺ cell (p=7.89×10⁻³) than was observed in patients at early stages(B, C) of the disease (FIG. 2). No significant correlation was foundbetween PLF levels and the number of CD8⁺ cells. These results areconsistent with the idea that PLF is produced and secreted by CD4⁺cells.

In contrast, a negative correlation (coefficient of -0.3, p<0.0001) wasfound between the concentration of normal ferritin and the number ofboth CD4⁺ and CD8⁺ lymphocytes. The increase in total ferritinparalleled the progressive lymphoid depletion, suggesting that theprincipal source of normal ferritin is not HIV infected lymphocytes.

7.2.3. CELL SURFACE ANTIGENS OF LYMPHOCYTES FROM HIV INFECTED PATIENTS

Since it was previously shown that PLF binds to T cells and blocks sheeperythrocyte rosettes (Moroz et al., 1977, Clin. Exptal. Immunol.29:30-35; Giller et al;, 1977, Cancer Immunol. Immunother. 3:101-105) weinvestigated the possibility that PLF bearing lymphocytes could beidentified in HIV infected patients. As shown in FIG. 3, 3-28% of thecirculating lymphocytes from HIV infected patients at various stages(A-E) reacted with CM-H9 McAb, and thus were exhibiting surface PLF. Asmall proportion (0.2-2.5%) of the lymphocytes also exhibitedcytoplasmic PLF (FIG. 3).

As expected, the T cell subset ratio was reversed in HIV infectedpatients (Table III).

                  TABLE III                                                       ______________________________________                                        CELL SURFACE ANTIGENS IN HIV INFECTED                                         PATIENTS AND NORMAL SUBJECTS                                                             Percentage of Lymphoid Cells                                                  Stained (mean ± SD)                                             Lymphoid     Healthy Donors                                                                            HIV-Infected.sup.a                                   Cells        (n = 35)    (n = 12)                                             ______________________________________                                        T4.sup.+     49.5 ± 9.4                                                                             24.7 ± 14.5                                       T8.sup.+     22.9 ± 6.3                                                                             39.8 ± 13.8                                       T11.sup.+    76.2 ± 9.7                                                                             81.8 ± 9.0                                        T11.sup.+ T4.sup.- T8.sup.-b                                                                6.9 ± 7.8                                                                             20.1 ± 8.2                                        PLF.sup.+c    0.78 ± 1.17                                                                           15.2 ± 6.4                                        ______________________________________                                         .sup.a Number of HIVinfected patients at the following stages: A = 1, B =     2, C = 6, D = 1, E = 2.                                                       .sup.b Percentage of T11.sup.+ cells less the sum of the percentages of       T4.sup.+ and T8.sup.+ cells. The results shown differ significantly as        measured by the Student's Ttest (p = 9.76 × 10.sup.-6).                 .sup. c p < 10.sup.6. For PLF staining, 15 normal donors of the 35 donors     used for T cell subset quantitation were analyzed. For all HIVinfected        patients, all membrane markers (including PLF) were assayed                   simultaneously.                                                          

Lymphocytes identified by McAbs T4 and/or T8, accounted for asignificantly smaller proportion of those stained by T11 McAb in HIVinfected patients compared to normal subjects (Table III). Thisobservation revealed the existence of an expanded T11⁺ population whichdid not react with either T4 or T8 monoclonal antibody (T11⁺ T4⁻ T8⁻).This population is significantly higher (p=9.76×10⁻¹⁰) in HIV infectedpatients than in normal controls (6.9±7.8%). This subpopulation (T11⁺T4⁻ T8⁻) in HIV infected patients (where it is present at approximately13.2% more than in normal subjects) is similar to the size of thepopulation identified as PLF positive (15.16±6.39%) (Table III) in thesame HIV-infected subjects.

Further experiments were carried out to elucidate which of the T cellsubsets bore surface PLF reactive with the CM-H9 McAb. Dual labeling forCD4 and PLF on lymphocytes from two subjects with late stage HIVinfection (Table IV), indicated that PLF is associated mainly withnon-CD4⁺ lymphocytes.

                  TABLE IV                                                        ______________________________________                                        DOUBLE MEMBRANE IMMUNOSTAINING OF                                             LYMPHOCYTES FROM AIDS PATIENTS USING ANI-T4,                                  AND ANTI-PLF (CM-H9) MONOCLONAL ANTIBODIES                                                 PERCENTAGE                                                                    LYMPHOCYTES STAINED                                              Lymphocyte     Patient No. 1                                                                            Patient No. 2                                       ______________________________________                                        T4.sup.+       42         28                                                  PLF.sup.+      13         13                                                  T4.sup.+ PLF.sup.+                                                                            0          5                                                  NOT STAINED    48         54                                                  ______________________________________                                    

7.2.4. THE EFFECT OF LEVAMISOLE ON CELL SURFACE ANTIGENS OF LYMPHOCYTESFROM HIV INFECTED PATIENTS

When the peripheral blood lymphocytes of HIV infected patients weretreated in vitro with levamisole, two concomitant phenomena occurred.The number of cells stained with the T8 McAb increased by about 20%(FIG. 4A), while the number of CM-H9 positive lymphocytes (PLF coated)decreased by about 15% (FIG. 4B). The number of T4⁺ stained cells didnot change following levamisole treatment (FIG. 4A). The number of T11⁺(CD2⁺) cells also did not change following levamisole treatment. Theseresults, taken together with the results obtained following duallabeling with the CD4 and PLF specific McAb, suggest that levamisolecaused shedding of membrane bound PLF, unmasking CD8 determinants on a

proportion of CD8⁺ CD2⁺ T cells. Parallel incubation in tissue culturemedium did not have an equivalent effect (FIG. 4A and 4B).

8. ISOFERRITINS IN PATIENTS WITH LYMPHOPROLIFERATIVE DISEASES

In the examples detailed below, serum levels of total ferritin and PLFwere measured in healthy individuals and in patients withlymphoproliferative disease and multiple myeloma. The majority of normalsubjects were deficient in PLF in the serum. Increased serum levels ofPLF were observed in patients with Hodgkin's lymphoma and non-Hodgkin'slymphoma of low and intermediate grades, as well as in patients withacute lymphocytic leukemia (ALL). Total ferritin was also elevated inthese patients. Chronic lymphocytic leukemia (CLL) and multiple myelomapatients exhibited normal levels of common serum ferritin, whereas PLFin the serum was lacking (See also, Moroz et al., 1987, Exp. Hematol.15:258-262).

8.1. MATERIALS AND METHODS 8.1.1. SUBJECTS

Serum samples were obtained from 40 blood bank donors who werehematologically normal, and from 70 patients with variouslymphoproliferative disorders, as well as from patients with multiplemyeloma. There were 20 patients with chronic lymphocytic leukemia (CLL),18 patients with non-Hodgkin's lymphoma, 15 with Hodgkin's disease, fivewith multiple myeloma, and two with ALL. Of the patients withnon-Hodgkin's lymphoma of intermediate grade, two had peripheral bloodinvolvement. A patient with non-Hodgkin's lymphoma and the two patientswith ALL received six packages of packed red blood cells each prior toferritin determinations. Serum samples were taken during one of thefollowup periods, at diagnosis, and during treatment or active disease.Only in patients with Hodgkin's disease was serum also taken duringremission. The classification of lymphoma was made according to aworking formulation (Krueger et al., 1983, Cancer 52:833).

8.1.2. MONOCLONAL ANTIBODIES

McAbs CM-G8 and CM-H9 were produced against human PLF as previouslydescribed in Section 6 et seq. supra, (see also, Moroz et al., 1985,Clin. Chim. Acta 148:111). McAbs were obtained from ascites fluid afterprecipitation with 50% saturated ammonium sulfate solution. The PLF usedfor the standard was obtained after purification on a diethylaminoethyl(DEAE)-cellulose column, as described above (see also, Moroz et al.,1985, Clin. Chim. Acta 148:111). Liver-ferritin standards were obtainedfrom MELISA ferritin kits (Elias Medizintechnik, Freiburg, FRG). Theamount of PLF that bound 2.50 pg of alkaline phosphatase (AP)-conjugatedCM-H9 McAb was considered to be 10 U of PLF.

8.1.3. QUANTITATIVE DETERMINATIONS OF FERRITIN

MELISA commercial ferritin kits were obtained from Elisa Medizintechnikand were used according to the manufacturer's instructions. In this kit,the binding of perioxidase-conjugated polyclonal antihuman liverferritin is measured.

8.1.4. MONOCLONAL ANTIBODY ELISA FOR PLF AND COMMON ISOFERRITINS

The enzyme linked immunosorbent assay (ELISA) of Engval and Perlmann(1972, J. Immunol. 109:129), with the modification of Voller et al.(1975, Lancet 1:426), was used in the formating of an ELISA formeasuring the serum liver ferritin and PLF isoforms (McELISA type A andMcELISA type B, reprectively). In both assays, the McAb CM-G8, whichbinds to all ferritins, was coupled to the solid phase. For the secondsite, McAb-enzyme-conjugate reaction, CM-G8 McAb, was used in McELISAtype A and CM-H9 McAb was used in McELISA type B.

The type A and B McELISAs were performed as follows: the wells ofmicrotiter plate were coated with 150 μl CM-G8 McAb (100 μg/mlphosphate-buffered saline [PBS], pH 7.2) and incubated overnight at 4°C. The plate was washed three times with PBS-Tween (PBS and 0.05% Tween20) and shaken dry.

Test sera (100 μl) diluted 1:2 with McELISA type A and 1:4 with McELISAtype B in PBS-Tween 0.025% were added in duplicate to the wells. Serumdiluent and ferritin standards were also added in duplicate. A serumsample with an elevated ferritin concentration was placed in the diluentto determine recovery at high dilution. The plates were incubated at 4°C. for 1 hour in McELISA A and overnight in McELISA B, washed threetimes with PBS-Tween, and 100 μl of AP-McAb conjugate (0.4 μg) was addedto each well. The plate was incubated for an additional 120 minutes atroom temperature and washed again three times. The enzyme substrate(p-nitrophenylphosphate, 1 mg/ml of diethanolamine buffer, pH 8.0, and0.5 mmol MgCl₂) was added and the reaction stopped after 10-30 minutesby the addition of 0.05 ml of 2M NaOH. The amount of colored product wasmeasured by absorbance at 405 nm.

8.2. RESULTS

The results described below demonstrate that serum PLF levels areelevated in patients with lymphoproliferative diseases such as acutelymphocyte leukemia, active Hodgkins lymphoma and non-Hodgkins lymphomaof low and intermediate grade.

8.2.1. EVALUATION OF LIVER FERRITIN STANDARD BY DIFFERENT ELISAs

Liver ferritin standards obtained from MELISA commercial kits wereassayed by the new McELISA type A and compared to those from thecommercial MELISA kit. The binding pattern of liver ferritin atconcentrations ranging from 15 to 500 ng/ml was similar in both assaysystems.

Serum samples containing low and high ferritin levels, supplied byMELISA kits, were assayed by the two systems. The concentrations offerritin at the low-control level were 70 ng/ml and 57 ng/ml in McELISAtype A and the MELISA kit, respectively. Both values were within therange specified by the MELISA manufacturers (40-70 ng/ml). Theconcentration of ferritin at the high-control level was 500 ng/ml asassayed by both systems (manufacturer's given range, 350-500 ng/ml).

In addition, correlations were made between the ferritin resultsobtained by the McELISA type A and MELISA assays: 22 sera from normaldonors, covering the range 10-150 ng/ml, and 21 sera from cancerpatients, covering the range 50-400 ng/ml, were tested. The correlationcoefficient for the normal range was 0.98, with the regression equationy=1.05±8.1; for the higher range it was 0.967, with y=1.37±21.09.

The results demonstrate that the McELISA type A, using AP-conjugatedCM-G8 McAb, is suitable for the quantitative determination of normallevels of liver-type ferritin in the serum. However, in high-rangeferritin determinations, higher quantities were measured by the McELISAtype A than by the MELISA.

8.2.2. BINDING OF PLACENTA AND LIVER FERRITINS TO CM-G8 AND CM-H9 MCABS

Both placenta and liver ferritins were measured in McELISA type A usinga conjugate of AP-CM-G-8 McAb. Similar patterns of binding of these twoisoferritins were observed at concentrations ranging from 30 to 800ng/ml. The results indicated that our newly developed McELISA type A issuitable for the measurement of both liver and PLF isoferritins. Bycontrast, a specific determination of PLF was possible only when theMcELISA type B was used.

The binding of AP-conjugated CM-H9 McAb to PLF in McELISA type B waslinear at concentrations ranging from 2.5 to 20 units, whereas liverferritin at concentrations ranging from 100 to 800 ng/ml did not bindAP-conjugated CM-H9 McAb. These results exhibit the specificity ofMcELISA type B for the detection of PLF.

8.2.3. ISOFERRITINS IN THE SERUM OF HEALTHY INDIVIDUALS AND PATIENTSWITH LYMPHOPROLIFERATIVE DISEASES

The results of the assay for isoferritins obtained from healthyindividuals and in patients with lymphoproliferative disease arepresented in Table V.

                  TABLE V                                                         ______________________________________                                        ISOFERRITINS IN HEALTHY INDIVIDUALS AND IN                                    PATIENTS WITH LYMPHOPROLIFERATIVE DISEASES                                    AND MULTIPLE MYELOMA                                                          Sources and                                                                   diagnosis    n      Ferritin (ng/ml)                                                                           PLF (U/ml)                                   ______________________________________                                        Blood bank   40      85.3 ± 65.9                                                                             8.1 ± 14.8                               donors                                                                        Male         24     108.0 ± 58.0                                                                             10.0 ± 10.0                              Female       16      50.3 ± 59.8                                                                             4.5 ± 7.7                                CLL.sup.a    20      66.3 ± 33.0                                                                             6.3 ± 13.5                               ALL.sup.b    2      600.0 ± 0**                                                                             140.0 ± 84.8*                             Multiple     5       68.5 ± 39.2                                                                             0                                           myeloma                                                                       Hodgkins lymphoma                                                             Active       5      359.0 ± 236.0**                                                                          47.0 ± 43.0*                             Remission    10      95.1 ± 46.7                                                                             15.3 ± 19.3                              Non-Hodgkin's                                                                 lymphoma                                                                      Low grade    7      218.3 ± 186.9*                                                                           97.1 ± 39.0**                            Intermediate 9      272.8 ± 180.8**                                                                          41.9 ± 35.8**                            High grade   2        380 ± 311                                                                                6 ± 8.5                                ______________________________________                                         .sup.a Chronic lymphocytic leukemia.                                          .sup.b Acute lymphocytic leukemia.                                            Significantly different from blood bank donors by Students ttest: *p <        0.025; **p < 0.005.                                                      

The mean concentration of ferritin measured in the sera of healthyindividuals by McELISA type A was 85.3±65.9 ng/ml (Table V). The meanferritin concentration was higher in males (108±58 ng/ml) than infemales (50.3±59.8 ng/ml) (Table V). Significantly higher ferritinlevels (p<0.025) were measured in the sera of patients suffering fromthe following malignant diseases: Hodgkin's lymphoma (359±236 ng/ml) andnon-Hodgkin's lymphoma of low and intermediate grades (218.3±186.9 and272.8±180.88 ng/ml, respectively), as well as in two patients with ALL(600±0 ng/ml). Sera of patients with Hodgkin's lymphoma in remissionshowed mean ferritin levels (95.1±46.7 ng/ml) similar to those ofhealthy individuals. Patients wtih CLL and multiple myeloma exhibitednormal ferritin levels (66.3±33 and 68.5±39.2 ng/ml, respectively). Theindividual ferritin concentrations measured with McELISA type A areshown in FIG. 5.

The mean serum concentration of PLF measured by McELISA type B in theserum of healthy individuals was 8.1±14.8 U/ml (Table V). Althoughhigher concentrations were measured in male 10±10 U/ml) than in female(4.5±7.7 U/ml) sera, these were not statistically significant. It isnoteworthy that 70% of the sera tested contained no detectable PLF.Elevated concentrations of PLF, significantly higher than normal(p<0.025), were measured in the sera of patients with Hodgkin's disease(47±43 ng/ml) and with non-Hodgkin's lymphoma of low and intermediategrades (97.1±39 and 41.9±5.8 U/ml, respectively). Patients with inremission had serum PLF levels not significantly different from those ofhealthy individuals (15.3±19.3 U/ml). High PLF levels were also measuredin two patients with ALL (range 80-200 U/ml). No or very low PLFconcentrations were found in the sera of patients with multiple myeloma(0) and CLL (6.3±13.5 U/ml), and 85% of the CLL patient's sera werecompletely negative. The individual distributions of PLF serumconcentrations are presented in FIG. 6.

9. ISOFERRITINS IN AUTOIMMUNE CONDITIONS

Serum levels of PLF were measured retrospectively from stored samplesderived from patients diagnosed with different autoimmune conditions.Results shown in FIG. 8 indicate that serum PLF levels were elevated inpatients diagnosed as having multiple sclerosis, myasthenia gravis, andrheumatoid arthritis. Each of these autoimmune conditions arecharacterized by immunodeficiency.

10. DEPOSIT OF HYBRIDOMAS

The following hybridomas have been deposited with the CollectionNationale de Cultures de Microorganisms of the Institute Pasteur inParis, France and received the accession number indicated:

    ______________________________________                                        Hybridoma     Accession No.                                                   ______________________________________                                        CM-H9         I-256                                                           ______________________________________                                    

The present invention is not to be limited in scope by the hybridomasdeposited or the embodiments disclosed in the examples which areintended as an illustration of one aspect of the invention, and anyhybridoma and method which are functionally equivalent are within thescope of this invention. Indeed, various modifications of the inventionin addition to those shown and described herein will become apparent tothose skilled in the art from the foregoing description. suchmodifications are intended to fall within the scope of the appendedclaims.

What is claimed is:
 1. A method for the prognosis and staging ofacquired immunodeficiency associated with HIV infection, comprising:(a)obtaining a serum sample and a peripheral blood lymphocyte sample from apatient; (b) measuring (i) the concentration of placental isoferritin inthe serum sample and, (ii) the number of CD4⁺ lymphocytes in theperipheral lymphocyte blood sample; and (c) determining the ratio of theconcentration of placental isoferritin in the serum sample to the numberof CD4⁺ lymphocytes in the peripheral blood lymphocyte sample, in which,an increase in the ratio indicates progression of disease.